THE AGE OF PURIFICATION
Figure 3. Process chromatography in 2006
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According to PubMed, over 10,000 articles with purification in the title were published in the decade between 1970 and 1980. This was the age of purification, enabled by an ever expanding
toolbox. A plethora of adsorbent alternatives for chromatography was developed, mostly based on dextran and agarose, but also
using cellulose, polyacrylamide, and methacrylates.25 In a 1990 review, Low26 discussed the methodologies available for scale-up, comparing their characteristics; these generally hold true today. Industry
pressure on high voume biopharmaceutical manufacturing is causing vendors to look for significant throughput and safety improvements.
Table 1. Characteristics of chromatographic methods of protein purification. Adapted from Low26 and Jungbauer.27
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It is significant that the number of biopharmaceutical products (biologics) was small before 1982, when recombinant human
insulin was approved. Products were generally purified from natural sources: human and animal blood, urine, pancreas, lung,
etc. Other than antibiotics, vaccines were essentially the only products of microbiology, which was in its infancy as a source
of product because only endogenously expressed proteins could be isolated. Biologics companies (and the regulatory agencies)
were generally focused on vaccines for protection against childhood infectious diseases and polio.28 Blood plasma products had been developed and introduced in response to a wartime need in early 1940s. Although there was
a renaissance of "industrial" methods and a search for simplicity in processing while maintaining safety, none of these biotherapeutics
were purified using the technologies commonly applied to biopharmaceutical production today. Early biologics differ significantly
from their microbiologically expressed successors as they were generally present at low concentration among many other proteins
from the same source. Thus purification problems were different from those of today, except where, for example, blood plasma
was used as source material from which multiple products are obtained.
Table 2. Biologics in the pre-recombinant DNA era. Adapted from tables compiled by Builder et al.29 * Note: most live attenuated vaccines in use today are derived from serial passage in cultured cells, including human diploid
cells (e.g., fetal lung tissue, other fibroblasts), monkey kidney cells, and chick embryos, among others. DPT = Diphtheria,
pertussis, tetanus; MMR = Measles, mumps, rubella.
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Marketed biologics in use before 1982 are shown in Table 2. Most, if not all, of these products have been withdrawn, substituted
by other products, or have been changed dramatically (and newly licensed). The states of purity and the formulations in which
they were first made available, not to mention the sources from which they are derived and the methods by which they are manufactured,
are significantly different today. For example, none of the products before 1960 were subject to purification schedules using
process chromatography. However, some products were made by local institutions or blood banks, which may have used rudimentary
purification on cellulose ion exchangers.
The introduction of chromatography in the early 1960s—mainly ion exchange and gel filtration—provided new opportunities for
purification, but the sources remained largely animal and human tissues (including blood) until the 1970s. During this period,
the focus in biochemistry was on purification as an enabling technology to improve the accuracy of structure and function
studies. Chromatography scale-up often was performed by a simple increase of column volume, with little regard to the maintenance
of column aspect ratios or residence time, and often restricted by the physical characteristics of the gels.
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